The presentation is detailed work on Glycyrrhiza glabra and Amaranthus tricolor with special focus on their hepatoprotective nature.

1. Pharmacognsy, Standardization
and Analytical work on
Glycyrrhiza glabra and
Amaranthus tricolor: The
Hepatoprotective Agents
PRESENTED BY:
GARIMA SHREE

2. LIVER
LIVER IS ONE OF THE MOST VITAL ORGAN IN HUMAN BODY.
 FUNDAMENTAL ROLES OF LIVER INCLUDES: METABOLISM, STORAGE,
DETOXIFICATION OF EXOGENOUS AND ENDOGENOUS SUBSTANCE.
OTHER ROLES INCLUDE: SUPPLY OF ENERGY, METABOLISM OF CARBOHYDRATES,
AND FATS, SECRETION OF BILE JUICE AND STORAGE OF VITAMINS.

3. FUNCTIONS OF LIVER
Biotransformation
•Ammonia
•Xenobiotics
•Haemoglobin
•Drugs
Synthesis
•Albumin
•Growth factor
•Urea
Storage
•Vitamins
•Glycogen
•Lipids

4. DAMAGE
INDUCING
AGENTS
FOR LIVER
Biological factor: Bacteria, Viruses, Parasites etc.
Autoimmune disorder: Immune hepatitis and
Primary bile cirrhosis
Toxic drugs: Excessive use of Alcohol, High dose
of Paracetamol, or anti tubercular drug etc
Toxic compounds: Carbon tetrachloride,
Thiacetamide

5. WHAT ARE
HEPATOPROTECTIVE?
 AS HEPATIC DISORDERS ARE INCREASING
SUBSTANTIALLY AND ACCOUNTS FOR 15% OF
WORLD’S BURDEN OF DISEASE, IT BECOMES
IMPORTANT FOR US TO USE HEPATOPROTECTIVE.
HEPATOPROTECTIVE OR ANTIHEPATOTOXIC
SUBSTANCE ARE THOSE AGENTS THAT HELPS IN
PROTECTION OF LIVER FROM VARIOUS DAMAGE
INDUCING SUBSTANCES THAT CAN ALTER THE
NORMAL FUNCTIONALITY OF LIVER.

6. HERBS AS HEPATOPROTECTIVE AGENTS
• THERE ARE VARIOUS HERBS PRESENTS IN OUR SURROUNDING THAT ACTS AS
HEPATOPROTECTIVE AGENT.
• IN CURRENT SCENARIO, NATURAL SUBSTANCES ARE PREFERABLE THAN
SYNTHETIC DRUGS FOR PRECAUTION TO IMPROVE IMMUNITY AND STRENGTHEN
ORGAN SYSTEM OF OUR BODY.
• VARIOUS HERBS THAT ARE USED AS HEPATOPROTECTIVE AGENTS ARE:
Plants Active constituent as
hepatoprotective
1. Vitis venifera Flavanoids like Catechins,
epicatechins, anthocyanidins etc
2. Opuntia fica-indica Cladodes
3. Glycyrrhiza glabra Glycyrrhizic acid
4. Amaranthus tricolor Polyphenolics, Flavanoids

7. GLYCYRRHIZA GLABRA

8. Taxonomica
l
classificatio
n:
Scientific name (Binomial nomenclature):
Glycyrrhiza glabra, Glycyrrhiza uralensis
Kingdom: Plantae
Subkingdom: Tracheobionta
Superdivision- Spermatophyta
Division: Magnoliophyta
Class: Magnoliopsida
Sub-class: Rosidae
Order: Fabales
Family: Fabaceae
Genus: Glycyrrhiza
Species: Glabra, Uralensis, inflata

9. LIQUORICE
Biological source: Liquorice is obtained from dried
peeled, unpeeled roots of Glycyrrhiza glabra,
belonging to the family Leguminosae.
Common name: Liquorice, Sweet wood, Yashti,
madhuh.
Geographical source: Liquorice is grown in the sub-
Himalayan tract.
It is mainly found in China, Europe, India, Iraq, Japan,
Kurdistan, Spain, Turkey, and the United States.

10. PHARMACOGNOSY
A. Morphology:
Perennial shrub
Height- 2-2.5 metres
Leaves: compound, Imparipinnate, alternate, 4-7 pairs oblong, elliptical, or lanceolate
leaflets.
Flower:Lavendar to violet colour, narrow, papilionaceous, borne in axillary spike.
Calyx: Short, campanulate, with lanceolet tips, glandular hairs.
Fruits: legume or pod, Upto 1.5 cm long, glabrous, reticulate pitted, 3-5 brown renniform
seed.
Roots: Taproots, of length upto 1.5 cm. Sudivided into subsidiary roots, 1.25cm, from this a
hard woody stolon arises. Which may reach upto 8 m.

11. B.
MACROSCOPICAL
CHARACTERISTICS
Macroscopic characteristics refer to the various character like Shape,
size, color, odour and special feature like touch and texture.
Color: Unpeeled liquorice – Externally yellowish, dark brown, and
internally Yellowish in colour.
Odor: Faint and characteristic
Taste: Sweet
Shape: Unpeeled drug: Straight and nearly cylindrical
Peeled drug: Mostly angular
Fracture: Fibrous in bark and Splintery in the wood.

12. C. MICROSCOPIC
CHARACTERISTIC
S:
Transverse section: Several Yellow-brown Cork layers
present. 1-3 cell layer thick phelloderm.
The cortex exhibits medullary rays, and obliterated sieve
portion radiate alternately.
Phloem consists of phloem fibres, and are surrounded by
crystal cells, with thick and incomplete lignified walls.
Vessels consist of Xylem fibres, surrounded by crystal cells
and parenchymal cells, where the parenchymal cells
contain of starch grain and single crystals of calcium
oxalate.

14. D. CHEMICAL
CONSTITUENT
S
There are more than 400
chemical constituents isolated
from Glycyrrhiza glabra.
Major biological component:
Glycosides, Flavonoids,
Polyphenols etc.

15. 1. Saponin:
The main Saponin in glycyrrhiza glabra is glycyrrhizin.
Glycyrrhizin is 60 times sweeter than cane sugar.
Other includes:
1. Glycyrrhizic acid is major triterpenoid saponin (4-
20%).
2. Glycirrheitic acid is the aglycon portion of glycoside.
3. Glucuronic acid
GLYCYRRHIZIN

16. Glycyrrhizin helps to lower serum liver enzyme levels, and improves tissue
pathology in hepatitis patients.
Glycyrrhizic acid improves liver histology and and induces the reduction of
serum aminotransferase.
Glycyrrhetinic acid was found to have protective effect against carbon
tetrachloride induced hepatotoxicity or retorsine induced hepatic damage.
Long-term use of glycyrrhizin has also been shown to prevent the
development of hepatocellular carcinoma in chronic hepatitis C patients

17. 2. Phenol content:
The main Phenol constituents include Liquiritin, isoliquiritin, iso-prenoid substituted
flavonoids, chromenes, coumarin, dihydrostilbenes.
Concentration: 7.45+0.05 mg/gm of Gallic Acid Equivalent (GAE).
3. Flavanoids:
More than 300 flavonoids were found in Glycyrrhiza glabra.
Most common flavonoids are: flavanones, isoflavanes, chalcones, isoflavenes.
The yellow color of glycyrrhiza is due to flavonoids, like liquiritin, isoliquiritin, etc.
Other liquorice flavonoid separated includes, rhamnolliuiritin, liquiritin apioside,
licoflavanone, isoliquiritigenin, licuraside, licochalcone A and B etc.
Several studies have also detected flavonoids including licoagrodin, licoagrochalcones,
glyinflanin B, and glycyrdione A.

18. LIQUIRITIN
FLAVANONES

19. 4. COUMARINS:
Liqcoumarin, glabrocoumarone A and B, herniarin, umbelliferone, and
glycyrin are all members of the glabra family.
G. glabra coumarins glycocoumarin and licopyranocoumarin prevented
the development of giant cells in HIV-infected cell cultures.
5. Essential Oils and Other compounds:
Fatty acids, phenol, guaiacol, asparagines, glucose,
sucrose, fructose, polysaccharides, and sterols (β-
sitosterol, dihydrostigmasterol) have also been
discovered as secondary metabolites.
Compounds such as alpha-pinene, octanol, stragole,
isofenchon, beta-caryophyllene, citronellyl acetate,
caryophyllene oxide, and geranyl hexanolate were discovered in
the essential oil of G. glabra.
COUMARIN

20. Mechanism Of Action of
liquorice:
•According to research, Secondary
metabolites extracted from licorice have
been found to lower serum liver enzyme
levels and boost tissue pathology in
hepatitis patients.
•When administered in a single dose of 2
mg/kg body weight per day, the
aqueous extract of G. glabra showed a
major effect in improving liver functions
in acute liver diseases.
•Glycyrrhizic acid greatly decreased
serum aminotransferases and enhanced
the histology of the liver.

21. Tradition
al Use:
Ganco was described in the Chinese medicine system
as an Antidote, to aches, a toxic substance.
Traditional use of Ganco was by preparing of
decoction of its dried roots and plants.
It is generally used in the treatment of acute and
chronic liver injury, viral hepatitis, hepatic steatosis,
Liver fibrosis, Hepatoma, Viral myocarditis.
In human body glycyrrhizin acid metabolizes to
glycirrhitinic acid, therefore both shares same
pharmacological activity.
It shows antioxidant, antifungal, anticarcinogenic,
anti-inflammatory, and cytotoxic activities.

22. Standardization
of
Glycyrrhiza
glabra

23. • The low amount of total
ash, acid insoluble ash and
water-soluble ash indicates
that absence of the
impurities in the crude
drug.
• The moisture contents in
the extract of g. glabra
roots was found to be 0.56,
hence indicating that the
drug was properly dried and
well stored.
PHYSICOCHEMICAL
PARAMETER
AVERAGE VALUE +/-
MEAN
Total Ash value 4.67 ± 0.35%
Acid Insoluble Ash 0.56 ± 0.34%
Water Soluble Ash 6.54 ± 0.22%
Loss on Drying (LOD) 5.87 ± 0.65%
Moisture contents 0.56 ±0.054%

24. MICROSCOPICAL
EVALUATION
• POWDER MICROSCOPY IS
DONE USING POWDER
FORM OF STEM OF
GLYCYRRHIZA GLABRA
WHERE IT IS TREATED
WITH VARIOUS
REAGENTS TO TEST THE
PRESENCE OF TANNINS,
LIGNIFIED CELLS,
STARCH, CALCIUM
OXALATE CRYSTAL.
S.no Reagents Observatio
ns
Characteristi
cs
1.
Phloroglucin
ol + conc.
HCL
Red Lignified
cells
2.
Fecl3
solution
Dark
blue to
black
Tannin
cells
3.
Iodine
solution
Blue Starch
4.
Phloroglucin
ol + conc.
HCL
dissolve
d
Calcium
Oxalate
crystals

27. HISTOPATHOLOGICAL
EVALUATION
• IN STEM TRANSVERSE SECTION:
• WELL-DIFFERENTIATED CORTEX AND PERICYCLIC FIBERS DISTRIBUTED
THROUGHOUT.
• PHLOEM, XYLEM, AND A WELL-DEFINED CENTRAL PITH .
• THE STEM'S T.S. CONSISTS OF SEVERAL ROWS OF THIN TANGENTIAL
ELONGATED CELLS ON THE OUTER CORK.
• THE CORTEX IS A LARGE ZONE OF THE STEM SECTION MADE UP
ENTIRELY OF PARENCHYMA CELLS WITH NO INTERCELLULAR SPACES.
• PERICYCLIC FIBRES ARE ORGANIZED IN A CIRCULAR PATTERN IN THE
CORTEX.

28. • VASCULAR BUNDLES OF FIBRES AND SEIVE
COMPONENTS ARRANGED RADIALLY ABOVE THE
XYLEM AND LOCATED ABOVE THE XYLEM.
• THE XYLEM TAKES UP THE MAJORITY OF THE
STEM. XYLEM IS MADE UP OF 2-3 COMPONENT
CELLS WITH WELL-DIFFERENTIATED PARENCHYMA
AND XYLEM FIBRES.
• THE MEDULLARY RAYS SPLIT THE RADIALLY
ORGANISED XYLEM.
• MEDULLARY RAYS ARE UNISERIATE TO
MULTISERIATE, TANGENTIALLY ELONGATED CELLS
THAT STRETCH FROM THE CENTRE REGION UP TO
THE INNER CORTICAL REGION.

29. PHYTOCHEMIC
AL SCREENING
I. METHOD OF EXTRACTION:
Leaves of Liquorice were collected, washed and powdered. 160 grams powdered leaved
was uniformly packed for Soxhlet apparatus.
It is extracted with ethanol and distilled water as solvent. Extraction was carried out for
20 hrs.
After extraction, the extract was filtered in Whatman filter paper, while hot in order to
remove any impurities.
Vacuum distillation was used to concentrate the residue
The volume was reduced to tenth of its volume.
The residual solvent was evaporated in water bath and dessicator.
Dried extract was packed in air-tight container.

30. TEST METHOD OBSERVATION RESULTS
1. ALKALOIDS Solvent free extract + Hcl a. Maeyer’s reagent: Cream
ppt
b. Hager’s reagent: Yellow
ppt
c. Wagner’s reagent: Reddish
brown ppt
d. Dragondroff’s reagent:
Orange brown ppt
a. Alkaloids were
present.
2. CARBOHYDRATE
AND GLYCOSIDES
Small amount of extract with 4ml
distilled water and filtered
A. Molisch test
B. Legal’s test
C. Borntrager’s test
a. Molisch test: appearance
of brown ring at
intersection of two liquid
b. Legal test: Pink to Red hue
c. Borntrager’s test:
Ammonia layer turns pink
a. Carbohydrates
were present.
b. Glycosides were
present.
3. PHYTOSTEROLS The extract was heated in a
solution of alcoholic potassium
hydroxide. Ethyl ether was used
to dilute the mixture and remove
it. The ether layer was
evaporated, and the residue was
analysed for phytosterol.
a. Lieberman Buchard test:
Presence of a bluish green
hue.
a. Phytosterols
were present.

31. TEST METHODS OBSERVATION RESULTS
4. GUMS AND
MUCILAGE
Small amounts of the
extract were added
separately to 25 ml of
absolute alcohol and
filtered while constantly
stirring.
The precipitate was
dried in the air and
tested for the presence
of gums and mucilage,
as well as its swelling
properties.
Gums and Mucilage were
absent in Glycyrrhiza
extract
5. SAPONINS Ethanolic extract was
diluted in 20 ml distilled
water and agitated for 15
minutes.
Saponins were detected
in the formation of a 1
cm layer of foam.
Saponins were present.
6. PROTEINS AND
AMINO ACID
The extract was
dissolved in a few ml of
water and then treated
with the reagents
a. Millon’s reagent: no
appearance of red
colour
b. . Ninhydrin reagent:
No purple-violet
colour
c. Biuret test: negative
Amino acids and Proteins
were absent.

32. TESTS METHODS OBSERVATIONS RESULT
7. PHENOLIC COMPUNDS
AND TANNINS
Small amounts of the
extract were separated
in water and tested.
a. Dil. Ferric chloride (5%) :
no Violet colour
b. 1% solution of gelatin
containing 10% sodium
chloride- no white ppt
c. 10% lead acetate
solution- no White ppt
a. Phenols are absent
b. Tannins are absent
8. FLAVANOIDS Extract are tested for
different substances.
Shinoda’s test:
Small amounts of the extract
were dissolved in alcohol,
then a piece of magnesium
was added, accompanied by
drops of Conc. hydrochloric
acid, and the mixture was
heated.
1. With NaOH- Blue to violet
colour
2. With H2SO4: : Yellow
orange color and orange
to crimson (flavonones).
3. Shinoda test: Magenta
colour
a. Flavanoids are present

33. ANALYTICAL WORK

34. 1. GLYCYRRHIZIC ACID:
Glycyrrhizic acid is the main bioactive component of Glycyrrhiza glabra.
Literature survey revealed only one analytical method for the estimation of
GA from extract that has been properly substantiated by a mass
spectroscopic analysis.
However, several chromatographic method of estimation has been
reported.
LC- DAD method for quantification and LC MS-MS system with gradient
elution for identity confirmation.

35. LC- DAD and LC-MS/MS analysis:
Instrumentati
on:
Binary Gradient system
Separation was caused in reverse phase column.
The mobile phase was a mixture of 65%, 5.3 mM
phosphate buffer (pH = 3.0) and 35% acetonitrile .
The flow rate was 1.0 ml/minute and the column
was maintained at ambient temperature.
The column effluent was monitored at 252 nm with
PDA detector.
LC-MS/MS analysis a different chromatographic
condition was maintained.
Preparation
of standard
solution
and Sample
solution:
Stock solution was prepared using 15.5 mg of
glycyrrhizic acid mono-ammonium salt in 25
ml of hot water.
The stock solution was diluted to the range
124 μg/ml to 12.4 μg/ml of GA for analysis.
Six point calibration curve was drawn for
linearity study and quantification purpose.

36. Validation:
•The analytical method was validated as per USP and ICH guidelines.
•To ensure reliability and accuracy of the method, recovery studies were carried out by mixing a known quantity of the
standard drug with the sample at three different concentration levels (10, 20 and 30% above assay value labeled as A, B and
C).
•Six injections of the standard solutions were done to study the precision of the method.
•The linearity of the method was established by triplicate injections of standard solution in the concentration range of 12.4
to124 μg/ml.
•The specificity of the method was studied from purity plot of PDA detector.
•The limit of detection (LOD) and the limit of quantitation (LOQ) were determined by injecting progressively low
concentrations of standard solution under optimized chromatographic conditions
Statistical Analysis: The statistical analysis was carried out on Sigma plot software (Version 8.02 SPSS
Inc., USA) and MS Excel 2007.

37. RESULTS
• THE MAXIMA IN CASE OF LC-DAD
ANALYSIS WAS OBSERVED AT 252 NM.
• THE LC-DAD ANALYSIS REVEALED THE
PEAK TO BE PURE AND SPECTRALLY
HOMOGENOUS WITH PEAK PURITY
ANGLE 0.357.
• THE AVERAGE RETENTION TIME OF GA
PEAK WAS 8.5 ± 0.09 MINUTES (±
S.D.; N=3).
• IN CASE LC-MS/MS ANALYSIS THE MASS
SPECTRUM REPRESENTED A HIGH
DEGREE OF IDENTITY CONFIRMATION.
• THE PRESENCE OF MOLECULAR ION
PEAK AT M/Z = 839 FOR BOTH THE
SAMPLE AND THE STANDARD
SPECTRUM WAS TAKEN AS
CONFIRMATION THAT THE PEAK
OBSERVED WAS OF GA IN BOTH SAMPLE
AND STANDARD SOLUTIONS.

38. Graph between Au (Absorbance) vs minutes
A. HPLC chromatogram of pure GA
B. UV spectrum of GA obtained from LC-DAD analysis.

39. HPTLC
FINGERPRINTIN
G OF
METHANOLIC
EXTRACT OF G.
GLABRA
METHANOLIC EXTRACT:
• Cleaned roots were taken for the extract purpose
• Roots were dried below 60 degree Celsius,
powdered and passed through 14 sieve to obtain
uniform size.
• The dried powder ( 500g) was used for
continuous hot extraction, with Methanol.
• The dried powdered material was placed in
Soxhlet apparatus, on water bath for 6 hrs.
• The extracts so obtained were filtered and dried
with the help of rotary evaporator (Rotavapor-R-
210).
• The final polar extract kept at low temperature
for further investigations.

40. METHOD
S:
The solvent system of hexane: ethyl acetate: methanol (9:1:1) was
taken.
Air-conditioned room of temperature of 22°c and 55% humidity.
The methanolic extract (5 µl) was spotted on the pre-coated silica
gel 60F254 hptlc aluminum plates as bands of 6 mm width with the
help of the autosampler fitted with a 100 µl hamilton syringe.
The hexane: ethyl acetate: methanol (9:1:1) solvent system was
transferred to camag twin trough plate development chamber
lined with filter paper and pre-saturated with mobile phase (30
ml).
The resulted plates were air dried and scanned.
A spectrodensitometer (scanner 3, camag) equipped with ‘win cats’
planar chromatography manager (version1.3.0) software was
employed for the densitometry measurements, spectra recording
and data processing.
Absorption/remission was then measured at a scan speed of 20
mm/s.
Chromatograms were recorded at 254 and 366 nm.

41. RESULTS
• A TOTAL NUMBER OF 11 PEAKS AT DIFFERENT RF VALUES AND PEAK AREA AT 366 NM
WERE OBSERVED IN THE HPTLC CHROMATOGRAMS .
HPTLC Photograph of methanolic
extract of Glycyrrhiza glabra at
366 nm. Chromatogram of the methanolic extract of Glycyrrhiza
glabra at 366nm.

42. • 10 peaks were observed in HPTLC chromatogram at 254 nm.
HPTLC Of methanolic extract of
Glycyrrhiza glabra at 254nm
Chromatogram of methanolic extract of Glycyrrhiza glabra at 254
nm.

43. Wave
length
Solvent system No. of peak Rf value Percentage peak area
366 nm Hexane: Ethyl
acetate:
Methanol
(9:1:1)
11 0.09,0.22,0.30,0.35,0.42,
0.46,0.54,0.60,
0.75,0.83,0.93
19.90,5.56,13.44,5.07,17.62,3.40,
8.24 ,2.12,17.37,4.88,2.40
256 nm Hexane: Ethyl
acetate:
Methanol
(9:1:1)
10 0.05 ,0.19, 0.35, 0.47
0.65, 0.79, 0.92, 0.97,
0.99 ,0.93
2.67, 3.56, 3.47, 7.79, 6.51, 8.95,
59.28, 5.09, 4.19, 5.83
Conclusion:
The fingerprint images of G. glabra roots developed from these HPTLC study might be referred to as
the standard reference fingerprints. These fingerprint images can be used for identification,
authentication, purification, and to separate G. glabra roots from its adulterants for ensuring
therapeutic efficacy.

44. Amaranthus
tricolor

45. TAXONOMICAL CLASSIFICATION
Domain: Eukaryota
Kingdom: Plantae
Phylum: Spermatophyta
Subphylum: Angiospermae
Class: Dicotyledons
Order: Caryophyllale
Family: Amaranthaceae
Genus: Amaranthus
Species: tricolor

46. Amaranth
us
tricolor
Biological source: It is obtained from leaves of Amaranthus tricolor
belonging to family Amaranthaceae. It is commonly known as
Laalshak, in India.
Geographical source: The species is found to be grown in Benin,
Nigeria, Kenya, Tanzania, and Southern Africa and throughout
India.
Climate and Soil: Amaranthus can be found in both tropical and
subtropical climates. Leaf amaranth is a warm-season crop that
thrives in mild, humid climates
The ideal pH range is 5.5-7.5, but some forms can thrive in soils
with pH levels as high as 10.0.
The colour production of red amaranth necessitates direct
sunlight.
Amaranth thrives in well-drained loamy soil with plenty of organic
matter.

47. PHARMACOGNOSY
• A. MORPHOLOGY:
• AMARANTHUS IS AN ANNUAL HERB WITH ERECT GROWTH
AND SCARCE TO PROFUSELY BRANCHING HABIT.
• ROOTS: SHALLOW TO DEEP TAP ROOT SYSTEM.
• STEM: STEM ARE GREEN TO PURPLE OR WITH MIXED SHADES
OF THESE TWO COLORS GLABROUS AND SUCCULENT
• LEAF: LEAVES ARE SIMPLE, ALTERNATE. TIPS ARE GREEN TO
PURPLE OR WITH SHADES OF THESE TWO COLORS, PURPLE
COLORATION PROMINENT IN YOUNG LEAVES AND FADES
AWAY AT MATURITY.

48. Inflorescence: It is terminal and axillary branched
spikes differently colored from green to deep
purple to orange.
Flowers: Flowers are small, regular mostly
unisexual, monoecious, pentamerous,
membranaceous, often with persistent stamens
2-5, placed opposite to perianth parts, often
some reduced to staminoides, filaments either
free or united below, anther one or two to three
styles and stigma, carpels 2-3, syncarpous, ovary
superior with one cambylotropus ovule, styles
single to 2 to 3 branched

49. B. Macroscopy :
The roots of Amaranthus tricolor Linn. has a
creamish brown hue, an indistinct odor, a
cylindrical shape, and a fibrous fracture with a
thickness of 0.5-1 cm and a length of 10-12
cm. There were also a few secondary roots and
numerous rootlets with tapering ends that
rooted at nodules on the roots.

50. C. Microscopy of Roots of
Amaranthus tricolor
Vascular
Bundles
Pith
Cortex
Cork Cells
Transverse Section of roots
showing Vessels and fibres
1.
2.
Transverse section of roots
of Amaranthus tricolor

51. 4.
3
.
5
.
Powder microscopy showing
pitted vessels
Powder microscopy showing
xylem and phloem
Powder microscopy
showing cork cells

52. D.
Chemical
Constituen
ts
Betaxanthin and betacyanins are two pigments under betalins,
which are major pigments in A. Tricolor.
Two chlorophyll breakdown products were extracted, 132-hydroxy-
(132-s)-phaeophytin-a and chlorophyll b methoxylactone.
Linoleic acid (49%) and linolenic acid (42%) are the main unsaturated
fatty acids.
Palmitic acid (18-25% of total fatty acids) is the major saturated
fatty acid in seeds, stems, and leaves.
Linolenic, lignoceric and arachidic present in trace amounts.
Sterols and spinasterol was present in highest amount and 24-
methylenecycloartenol was found in the seeds only.
Betalins

53. Total phenolic content: Hydroxybenzoic acids -
Gallic acid (3,4-5Trihydroxybenzoic acid), vanilic
acid (4-Hydroxy-3-methoxybenzoic acid), Syringic
acid (4-Hydroxy-3,5-dimethoxybenzoic acid), p-
hydroxybenzoic acid (4-hyroxybenzoic acid),
Salicylic acid .
Flavanoids – Isoquercitin (quercetin-3-glucoside),
Rutin (quercetin-3-rutinoside), Isorhamnetin,
Kaempferol, quercitin
Gallic acid

54. TRADITIONAL
USES
Widely used in ayurveda and Siddha for treating menorrhagia,
diarrhea, dysentery, haemorrhages, cough, and bronchitis.
Externally, it's used as an emollient poultice or a mouthwash to
treat ulcerated throat and mouth conditions.
Coughs, throat infections, toothaches, eczema, piles, diarrhea,
gonorrhea, leucorrhea, and impotence are among the diseases
for which this plant is used.
The plant Decoction is used to enhance the liver and boost
vision. When given internally, root-paste combined with warm
water causes vomiting and purges toxic matter from the
intestine.

55. Standardization of
Amaranthus tricolor

56. PHYSICOCHEMICAL
PARAMETERS
Physicochemical
parameter RESULT (%)
Total Ash value 12.8
Acid Insoluble Ash 6.89
Water Soluble Ash 5
Loss on Drying (LOD) 6.7
Ethanol soluble extractive
value
7.6
Water soluble extractive
value
20

57. TEST METHOD OBSERVATION RESULTS
1. ALKALOIDS Solvent free extract + Hcl a. Maeyer’s reagent: Cream
ppt
b. Hager’s reagent: Yellow
ppt
c. Wagner’s reagent: Reddish
brown ppt
d. Dragondroff’s reagent:
Orange brown ppt
a. Alkaloids were
present.
2. CARBOHYDRATE
AND GLYCOSIDES
Small amount of extract with 4ml
distilled water and filtered
A. Molisch test
B. Legal’s test
C. Borntrager’s test
a. Molisch test: appearance
of brown ring at
intersection of two liquid
b. Legal test: Pink to Red hue
c. Borntrager’s test:
Ammonia layer turns pink
a. Carbohydrates
were present.
b. Glycosides were
present.
3. PHYTOSTEROLS The extract was heated in a
solution of alcoholic potassium
hydroxide. Ethyl ether was used
to dilute the mixture and remove
it. The ether layer was
evaporated, and the residue was
analysed for phytosterol.
a. Lieberman Buchard test:
Presence of a bluish green
hue.
a. Phytosterols
were present.

58. TEST METHODS OBSERVATION RESULTS
4. GUMS AND
MUCILAGE
Small amounts of the
extract were added
separately to 25 ml of
absolute alcohol and
filtered while constantly
stirring.
The precipitate was
dried in the air and
tested for the presence
of gums and mucilage,
as well as its swelling
properties.
Gums and Mucilage were
absent in ethanol soluble
amaranthus extract
5. SAPONINS Ethanolic extract was
diluted in 20 ml distilled
water and agitated for 15
minutes.
Saponins were detected
in the formation of a 1
cm layer of foam.
Saponins were present.
6. PROTEINS AND
AMINO ACID
The extract was
dissolved in a few ml of
water and then treated
with the reagents
a. Millon’s reagent:
appearance of red
colour
b. Ninhydrin reagent:
purple-violet colour
c. Biuret test: positive
Amino acids and Proteins
were present.

59. TESTS METHODS OBSERVATIONS RESULT
7. PHENOLIC COMPUNDS
AND TANNINS
Small amounts of the
extract were separated
in water and tested.
a. Dil. Ferric chloride (5%) :
Violet colour
b. 1% solution of gelatin
containing 10% sodium
chloride- white ppt
c. 10% lead acetate
solution- White ppt
a. Phenols are present
b. Tannins are present
8. FLAVANOIDS Extract are tested for
different substances.
Shinoda’s test:
Small amounts of the extract
were dissolved in alcohol,
then a piece of magnesium
was added, accompanied by
drops of Conc. hydrochloric
acid, and the mixture was
heated.
1. With NaOH- Blue to violet
colour
2. With H2SO4: : Yellow
orange color and orange
to crimson (flavonones).
3. Shinoda test: Magenta
colour
a. Flavanoids are present

60. ANALYTICAL WORK

61. 1. HPTLC
FINGERPRINT
ANALYSIS OF
CHLOROFORM
EXTRACT OF
AMARANTHUS
TRICOLOR
TLC and HPTLC fingerprint methods are often
used to gain preliminary information about the
chemical composition of plant extracts and to
identify specific plant species.
The separation of natural compounds with
different selectivities, such as flavonoids,
phenolic acids, alkaloids, coumarins, and other
phytochemicals, is possible using
chromatographic systems.

62. Solvent system: toluene and ethyl acetate in ration of 7:3.
The CAMAG Linomat 5 applicator was used to apply the plant
extract samples.
There were four tracks in all, with ATC application volumes of 5
and 10 microliters.
The plates were formed in a linear ascending direction in a
CAMAG glass Twin Trough Chamber 20x10cm with a 7:3
toluene: ethyl acetate solvent method.
There were four tracks in all, each spaced 23.3 mm apart.

63. Detection: For evaporation of solvents,
developed plates were dried in a hot air
oven at 60°C for 5 minutes.
WIN CATS planer chromatography manager
software was used to record the retention
factor (Rf) values and fingerprint data.

64. • THE EXISTENCE OF FOURTEEN
(14) IN A. TRICOLOR EXTRACT
WAS DISCOVERED
• THE NUMBER OF
CONSTITUENT PEAKS AND RF
VALUES, AS WELL AS THE
MAXIMUM PERCENT AND
AREA PERCENT, ARE
SUMMARIZED AS FOLLOWS
• .
RESULTS

65. Chromatogram profile of
Amaranthus tricolor at 366nm
HPTLC Chromatogram of Amaranthus tricolor

66. CONCLUSIO
N
There's a good chance that the chosen ATC extracts contain a lot
of flavonoids.
The selected plants' HPTLC finger printing profiles displayed a high
degree of similarity in their components with previous studies
which stated the presence of flavonoid sulphate, flavon C and C-
/O glycosides, and aglycons in their root and aerial portions.
The HPTLC finger printing profile of the plant A. Tricolor revealed
that its chloroform extracts contain a significant number of active
phytoconstituents.
These findings can prove useful in the future when it comes to
herbal drug standardization.

67. TLC
fingerprint
profile of
Amaranthus
tricolor.
TLC Fingerprinting is an important analytical
technique for separating, identifying, and
estimating various groups of phyto-compounds.
Hepatoprotective, hypoglycemic, hypolipidemic,
and hematological activity were some identified
functions.
For TLC finger printing, petroleum ether,
chloroform, and ethanolic extracts of both the
selected herbs were used.
Flavonoids were detected using standard rutin.

68. Materials and methods: petroleum ether, chloroform, and
ethanolic extracts of both the selected herbs were used.
The stationary phase was silica gel G, and the mobile phase
was a mixture of solvent systems.
Flavonoids were detected using standard rutin.
The chromatographic chamber was run in a linear ascending
direction, and the detection was done in a UV chamber.

69. Sample preparation: Small amounts of Amaranthus tricolor sample
extracts were taken and dissolved in the solvents. Pet ether,
chloroform, and ethanol were the plant extracts chosen.
Standard rutin Preparation: In 1 mL methanol, 10 mg of pure rutin
was dissolved. This was used for spotting on the activated TLC
plates that had already been prepared.
Suitable solvent system:
A. ethyl acetate: butanol: acetic acid: water (80:10:5:5): For all
three extract, but none of them showed differentiation of extract.
B. , the solvent proportions in the method were changed to
methanol: chloroform (7:3) and a few drops of formic acid (to aid
good clear resolution).

70. RESULTS
Extract Petroleum Ether Chloroform
Number of spots 3 7
Rf values 0.89, 0.77, 0.43 0.96, 0.85, 0.66
0.52, 0.30, 0.22,
0.12
Diameter of spot (mm) 3.50, 4.00, 3.00 6.00, 15.00, 10.50,
8.50, 5.50, 3.00,
3.50
Colour of respective
spot
Red, Red, Pink Pink, Red, Red, Pink,
Blue, Pink, Blue
Rf values, Diameters, and colour respective spots of Amaranthus tricolor both extracts.

71. The number and diameter
of spots in Chloroform and
Pet. ether extracts TLC of A.
tricolor
The number and Rf. value
of spots in Chloroform
and Pet. ether extracts
TLC of A. tricolor
Separation of spots from
sample herbs extracts with
corresponding Rutin sample.

72. CONCLUSION
S
Spots corresponding to the pure Rutin sample spot in both physical
visual colour and Rf value were found in two of the four TLC plates,
chloroform extracts of A. tricolour, out of the separated components.
The findings of this study clearly demonstrated the existence of
various phytochemicals of therapeutic value in the sample herbs of
A. tricolour, such as alkaloids, glycosides, and flavonoids, as well as
those of food importance, such as carbohydrates, vitamins, and
proteins, which collectively justified the herbs' nutraceutical
potential.
Due to outstanding positive chemical tests and great TLC spot
resolution, chloroform extract of studied herbs remained the best
extract with appreciable result when compared to other solvents
extract.

73. SUMMAR
Y
Glycyrrhiza glabra:
In Glycyrrhiza glabra, Glycyrrhizin and Glycyrrhizic
acids were the main component that acts as
hepatoprotective agents. Glycyrrhizin helps to
lower serum liver enzyme levels and improves
tissue pathology in hepatitis patients and
glycyrrhizic acid helps to improve liver histology.
The analytical technique confirms the presence of
the same and this also explains the traditional use
of licorice which was used in Chinese system of
medicine for its hepatoprotective nature and other
uses includes to use decoction of drugs to cure
aches, and the treatment of acute and chronic liver
injury, viral hepatitis, hepatic steatosis, liver

74. Amaranthus tricolor:
Amaranthus tricolor contains a lot of
flavonoids and polyphenolic compound that
helps in hepatoprotective activity. It was used
traditionally for its hepatoprotective nature, to
enhance the liver activity.
It is also known that the anti-oxidant property
of the herb may contributes to the
hepatoprotective activity of the herb.
Therefore, through analytical work,
polyphenolic compounds and flavonoids were
confirmed which were the main constituents to
contribute to hepatoprotective nature of the
plants.

75. Reference:
• HEMRAJ VASHIST*, DIKSHA SHARMA, PHARMACOGNOSTICAL
ASPECTS OF GLYCYRRHIZA GLABRA,NNOVARE ACADEMICS, Vol 6,
Issue 4, 2013, PAGE 56-58.
• Nazim A. Mamedov and Dilfuza Egamberdieva, Phytochemical
Constituents and Pharmacological Effects of Licorice: A Review,
Plant and Human Health, Volume 3, 2019 Feb 12 : 1–21, Published
online 2019 Feb 12.
• Asif Husain , Aftab Ahmad , Mohd Mujeeb , Shah Alam Khan , Afnan
Ghormulla Alghamdi , Firoz Anwar, Quantitative Analysis of Total
Phenolic, Flavonoid Contents and HPTLC Fingerprinting for
Standardization of Glycyrrhiza glabra Linn. Roots; iMedPub Journals;
2015 Vol. 1 No. 1:1, pg no. 1-9.
• Rajani Srivastav, An updated review on phyto-pharmacological and
pharmacognostical profile of Amaranthus tricolor: A herb of
nutraceutical potentials, Volume 6(6), The Pharma Innovation
Journal 2017, page no. 124-129.
• Prof Dr Ali Esmail Al-Snafi, Glycyrrhiza glabra: A phytochemical and
pharmacological review, Volume 8, Issue 6 version, IOSR Journal of

76. Reference:
• Jae-Chul Jung, Yun-Hee Lee, Sou Hyun Kim,3 Keuk-Jun Kim,4 Kyung-
Mi Kim,1 Seikwan Oh,5 and Young-Suk Jung, Hepatoprotective effect
of licorice, the root of Glycyrrhiza uralensis Fischer, in alcohol-
induced fatty liver disease, BMC complemnetary medicine and
therapy, Published online 2016.
• Gaber El-Saber Batiha, Amany Magdy Beshbishy, Amany El-
Mleeh,3 Mohamed M. Abdel-Daim, and Hari Prasad Devkota,
Traditional Uses, Bioactive Chemical Constituents, and
Pharmacological and Toxicological Activities of Glycyrrhiza glabra L.
(Fabaceae), Biomolecules. 2020 Mar, Published online.
• Umma Khair Salma Khanam and Shinya Oba, Bioactive substances in
leaves of two amaranth species, Amaranthus tricolor and A.
hypochondriacus, volume 93, Canadian Journal of Plant Science • 1
January 2013

77. THANK YOU!!